Worldwide, enteropathogenic E. coil (EPEC) and enterohemorrhagic E. coil (EHEC) are lethal human pathogens and are classified as Category B by NIAID. Studies on EPEC and EHEC pathogenesis are limited by an extremely complex genome, comprising 1387 gains and 528 losses compared to E. coil K12, and by a lack of functional assays for many of the proposed virulence factors. We have developed a model for EPEC and EHEC virulence using the nematode C. elegans, which will define novel virulence genes and host targets required for mammalian pathogenesis. Aim 1. EPEC and EHEC strains kill C. elegans under certain conditions, whereas commensal E. coil strains (K12) do not. Killing depends on genes in the LEE pathogenicity island, which are required for EPEC and EHEC pathogenesis in humans. By expressing the LEE in E. coil K12, we will determine whether genes in the LEE are sufficient to cause worm killing, or whether additional genes are required. Aim 2. The tryptophan metabolite indole is secreted and sensed extracellularly by E. coil and regulates stationary phase gene expression. Tryptophan is required for EPEC and EHEC to kill C. elegans. We will determine (i) whether the factor(s) that cause worm killing are secreted by EPEC or require ingestion by the worms; (ii) whether indole contributes directly or indirectly to C. elegans killing; and (iii) whether indole regulates the expression of EPEC and EHEC virulence factors. Aim 3. We will screen large numbers of mutant C. elegans screen for homozygous mutations that confer resistance to killing by EPEC and EHEC. After placement into complementation groups, the precise genes involved will be identified. We will also screen for EPEC and EHEC mutants that fail to kill C. elegans. Together, these studies should yield important information about mechanisms of virulence for these important human pathogens that has not been accessible by traditional experimental approaches. We anticipate that the data generated during the proposed funding period will serve as the basis for an R01 application.